The present invention relates generally to the field of chemical and microbiological assay techniques and more specifically to a new and improved method of preparing a blood sample for cell classification and enumeration.
Since the beginning of immunological research, researchers have desired to be able to enumerate, identify and analyze specific particles in biological fluids. In particular, bodily fluids of mammalian patients are often analyzed to enumerate target cellular components. For example, blood may be analyzed to determine the absolute count of T-lymphocytes per unit of volume of whole blood that positively express the CD4 or CD8 surface antigens. A CD4/CD8 blood test is important to determine the progression of the human immunodeficiency virus (HIV) throughout different stages of development and particularly for the diagnosis of acute development of the viral infection to Acquired Immune Deficiency Syndrome (AIDS). The quantity of testing required for diagnosis of the AIDS virus alone is fueling the demand for simplified, efficient methods for cell enumeration.
To accomplish this objective, researchers have developed methods to mark specific cells with fluorescent markers that combine with a cell according to a specific cell traits or characteristics. Early immunologists used fluorescent dyes to stain the nucleus of white blood cells. The fluorescence from the dye or stain made it easier to enumerate and identify white blood cells. The breakthrough of monoclonal antibody technology soon permitted the conjugation of a monoclonal antibody with a fluorescent dye. This expanded the immunologist's ability to enumerate and classify cells according to their surface antigens.
The development of light detection devices capable of making highly accurate quantitative measurements of fluorescent intensity, a new potential for automated cell enumeration devices emerged. Flow cytometers were developed with fluorescent sensors to detect fluorescent emission. scanning devices were developed to scan microscope slides and automatically identify and enumerate target blood cells.
The development of such new instruments for automated fluorescent analysis has resulted in a demand for new and improved sample preparation and presentation techniques that simplify greatly cell enumeration without jeopardizing accuracy. The general purpose of such techniques is to expand analytical capabilities, improve reliability, simplify preparation, minimize handling of samples, and reduce the risk of disease transmission during sample handling.
Flow cytometers have the advantage of making rapid and accurate cell enumeration of different cells in a sample, but does not make direct accurate enumeration of cells per volume of whole blood. Flow cytometers present a sample of cells or particles before a light source in a linear flow path to measure the interaction of the laser with each cell or particle. The flow path consists of a downward flowing stream of liquid into which the cells are released one at a time into the center of the flow path.
The laser beam strikes each cell, causing the laser light to scatter in several directions. Light detectors are spatially oriented to detect such scatter. Fluorescent detectors can also be utilized to measure fluorescent emission from a cell. Statistical analysis of the data collected from the detectors can be used to characterize and enumerate the cells. The drawbacks of flow cytometry include undesirable consequences that relate to both how a sample is prepared and how the sample is presented. First, the flow cytometry instrument requires a sequence of sample preparation steps to remove red blood and plasma components and fluorescent markers. The steps often damage the cells, particularly unhealthy cells that are often brittle. The margin of error of the total sample preparation technique is compounded by each handling step or statistical enumeration technique. Moreover, many steps such as centrifugation, filtration, lysing, cell washing, etc. cannot be accomplished without defeating, to a large extent, the ability to absolutely count particulate as a function of absolute volume.
For example, a common method of preparing a sample of blood for enumeration and classification of the white blood cells in the sample includes the steps lysing red blood cells and centrifuging the sample to remove the excess red blood cell debris from the plasma. Lysation ideally will destroy all of the red blood cells, and none of the white blood cells. However, the ideal is difficult to accomplish and the effectiveness of lysation varies from patient to patient. For example some patients have delicate white blood cells. The lysing agents may damage the delicate white blood cells. Other patients, particularly patients with AIDS, produce red blood cells with a heightened resistance to lysing agents. Such samples require multiple applications of lysing agents, more effective lysing agents which cause a greater threat of damage to white blood cells that are the subject or target of the cell enumeration assay.
Centrifugation or gravitational separation is likewise an imperfect separation method. When centrifugation is used with lysation the white blood cells are mixed with the fluid while the lysed cellular debris is centrifuged to the bottom of the centrifugation vessel. Then, the white blood cells are decanted from the debris. Unfortunately, the centrifugal separation is not perfect and some of the unlysed particles will remain mixed with the cellular debris. Whenever separation techniques such as centrifugation and decantation are involved, the precise amount of target cell per unit of volume cannot accurately be measured because of the error introduced by the separation techniques. For example, when a known volume of blood is centrifuged after lysing the red blood cells, the debris from the lysed red blood cells settles to a sedimentary layer. Then, the plasma containing white blood cells is decanted from the debris. Regardless of the care taken, some of the white blood cells will remain in the cuvette with the debris from the lysed red blood cells. Consequently, a subsequent count of the white blood cells separated from the debris would not be an accurate count of the number of white blood cells which were contained in the original sample.
Red blood cells are lysed because the presence of red blood cells creates optical interference when fluorescence is used to detect and enumerate a targeted subclass of cells. Two types of interference make red blood cells troublesome. First, red blood cells absorb light having a wavelength in the range of 200 nm to 500 nm. If laser light of fluorescent discharge occurs in this range, then the light that stimulates the fluorescent marker and the fluorescent discharge from the marker will be greatly weakened. Second, red blood cells and other plasma proteins autofluoresce These components of blood naturally contain fluorophores or molecules that discharge fluorescence when stimulated by light. Fluorescent markers must be chosen to have an energy of activation and an energy of fluorescent light discharged that likewise avoids autofluorescence of red blood cells.
Another drawback to flow cytometry techniques is that the sample is discharged from the nozzle in the form of atomized droplets, similar to a fine mist which can be carried through the air. Technicians using a flow cytometer may inhale or otherwise be exposed to the atomized particles. Should the sample be disease infected blood or other biohazardous material, then the technician is exposed to a risk of infection. Thus, there is a need for sample preparation methods to eliminate the risk of infection created by the release of these atomized particles.
Sample preparation for flow cytometers may include the use of fixatives or fixing agents which kill bacteria and viruses without damaging the physical structure of blood cells. Formaldehyde is a common fixative which may be added to the sample to destroy the infectious material. There are at least two drawbacks to the use of fixatives. First, fixing agents "kill the cell" without causing substantial damage to the physical structure. However, some morphological changes in the cell are caused by the fixative. Consequently, data from the fixed samples vary from data which would otherwise be obtained from untreated blood. Second, the addition of fixatives add a step of dilution. Each time a sample is mixed, handled, diluted, or centrifuged, a processing step is added to the overall procedure. The error of one step may not be significant alone; however, when the error is compounded with the error from each measurement step, the entire process may have a total undesirable margin of error. Consequently, the simplest possible assay technique with the fewest number of handling steps is advantageous to the researchers and practitioners that use fluorescent measurement devices because they make volumetric determinations more accurate.
Because volumetric enumeration (i.e., measurements of cells per unit of volume) is so important for diagnostic purposes, several attempts to make volumetric determination using a flow cytometer have been previously proposed. One common technique is to count all of the cells with a device such as a cell sorter to enumerate the total number of a certain subset of cells in a fixed volume of sample. Then, another sample is prepared that counts the number of target cells as a ratio of the cell type acting as a standard. Multiplying the ratio of target cells to standard cells by the number of standard cells per unit of volume gives a volumetric estimation of the number of target cells in a fixed volume. However, the margin of error from each cell enumeration is compounded together. The accuracy of volumetric cell counting devices can greatly be increased if the volumetric enumeration could be done in one measurement.
Another attempt to improve accuracy of volumetric cell enumeration for flow cytometry instruments entailed mixing a fixed number of fluorescent microparticles (e.g., beads) with a fixed volume of sample prior to the preparation techniques. Typically, the microparticles are labeled with the same fluorescent label as the cells targeted for enumeration. However, the concentration of markers on the microparticles is typically 5-10 times the intensity of the concentration of the labeled particles on the microparticles. A gate based upon the magnitude of fluorescent emission can be set to distinguish the microparticles from the target cells. Error is introduced into this technique when target cells have unusually high antibody concentration or when some of the microparticles have lowered fluorescent concentration. Even if the gating technique ensured that no microparticles would be confused with target particles, the technique requires two measurements to obtain one volumetric result. The error of the two measurement is compounded leaving the microparticle technique to be less accurate than a more direct technique that measures the number of cells per unit of volume in one measurement.
After the predetermined number of microparticles is mixed with the fixed volume of blood the sample is processed. During the processing step, some of the microparticles may be lost because the microparticles, which are typically made of polystyrene, have a density different from that of the target cells. Consequently, the sample is in continued need of mixing to ensure that the microparticles do not settle disproportionately.
Another known system for cell enumeration is fluorescent microscopy which combines fluorescent labeling with microscopy technology. Such systems include automated scanning microscopes to identify and enumerate subclasses of cells. Sample preparation includes smearing a microscope slide with a sample containing fluorescent stained or labeled blood cells (target cells). A light source is used to illuminate the cells against a grid in the background of the microscope optics. The number of cells per grid are counted and averaged to quantify the fluorescent stained cells. While this method can determine ratios of one cell type to another cell type, microscopic enumeration of smeared slides cannot determine directly the number of cells per unit of volume.
Furthermore, when manual cell counting is used, there is great opportunity for human error and fluctuations in accuracy from technician to technician. Automated techniques for counting cells smeared on a slide improves the ability to more accurately count the cells per unit of area across the slide, but methods of cell preparation that are compatible to the automated fluorescent microscopy cannot directly determine the number of target cells per unit of volume.
Beads likewise can be used with microscope slides to make volumetric enumeration of whole blood on a slide. One technique mixes substantially incompressible microparticles (e.g. beads) with a fixed volume of liquid sample before processing the sample and smearing on a slide. The ratio of target cells to microparticles times the number of microparticles per unit of volume can be used to estimate the number of target cells per unit of volume. However, this technique does not avoid the step of lysing. All of same problems of using microparticles with flow cytometry are equally applicable to the use of microparticles with fluorescent microscopy.
Consequently, there has arisen a need for a new and improved sample preparation and assay method that improves the accuracy and efficiency of volumetric cell enumeration and at the same time simplifies the preparation of the sample. Such a method would further be desirable if it eliminated separation and handling steps such as centrifugation, decantation, cell lysing, and cell washing. The assay would be especially beneficial if the volume of the sample could be preserved and accurately analyzed without need for microparticle additives or fixatives. The ability to analyze whole blood to make a precise volumetric cell identification and enumeration from either the entire sample or a portion of the sample would greatly expand and improve clinical and diagnostic applications of such assays techniques.